Characterization of small, mononuclear blood cells from salmon having high phagocytic capacity and ability to differentiate into dendritic like cells

Abstract

Phagocytes are the principal component of the innate immune system, playing a key role in the clearance of foreign particles that include potential pathogens. In vertebrates, both neutrophils and mononuclear cells like monocytes, macrophages and dendritic cells are all professional phagocytes. In teleosts, B-lymphocytes also have potent phagocytic ability. We have isolated a population of small (<5 µm), mononuclear blood cells from Atlantic salmon (Salmo salar L.) not previously characterized. In order to identify them, we have performed morphological, gene expression, flow cytometry, cytochemical, ultrastructural and functional analyses. Interestingly, they highly express the gene encoding CD83, the most characteristic cell surface marker for dendritic cells in mammals, and MHC class II limited to professional antigen presenting cells. They did not express genes nor did they have cell markers for B-cells, T-cells, monocytes/macrophages or neutrophils as shown by qRT-PCR, flow cytometry and immunoblotting. A remarkable feature of these cells is their potent phagocytic capacity. Their oxygen-independent killing mechanism, as shown by intense acid phosphatase staining, is supported by lack of respiratory burst and myeloperoxidase activity and the acid phosphatase's sensitivity to tartrate. They show a high level of morphological plasticity, as, upon stimulation with mitogens, they change morphology and obtain branching protrusions similarly to dendritic cells. We suggest, based on our findings, that the small, round cells described here are progenitor cells with potential to differentiate into dendritic like cells, although we can not exclude the possibility that they represent a novel cell type.

Figure 1. C4B6⁻ cells are small, round cells which are abundant among the PBL.

(A) Double immunostaining with anti-TO (green) and MAb C4B6 (red) revealed small, round cells that were C4B6⁻ (white arrows). (B) Flow cytometry histograms of PBL prior and after MACS separation. The bound fraction (C4B6⁺ cells) contained both polymorphonuclear (PML) and mononuclear cells (L = leukocytes, M = monocytes) while the cells in the unbound fraction (C4B6⁻ cells) were small, mononuclear cells with low level of granularity. Cells with large, round nuclei were present in most C4B6⁻ cells (labeled with *), although cells with other nuclei forms were observed. C4B6⁺ cells are shown as red dots in the dot plots (middle panels). The markers define the C4B6⁺ cells. Diff Quick stained cells are shown in the right panels. Representative single cells are enlarged. The diff quick pictures are captured with 63× objective. Scale bar = 5 µm.

Figure 2. The C4B6⁻ cells are negative for lymphocyte-, neutrophil and monocyte/macrophage markers, but express CD83 and MHC class II.

(A) Flow cytometry analyses of the unbound fraction after MACS show the C4B6⁻ cells' reactivity with MAb C4B6 (anti-leukocytes), C7G7 and G2H3 (B-cells), E3D9 (neutrophils) and the polyclonal anti-human CD3 antiserum (T- and IgM⁺ cells). Representative histograms are shown. Grey filled curves are negative controls. Positive cells are shown as red dots. The markers represent positive cells. The antibodies reactivity against PBL prior to MACS is shown in the lowest panels. (B) A silver stained SDS- polyacrylamide gel, left panel, and an immunoblot using polyclonal IgM serum developed with ECL, right panel. Lane 1, molecular mass markers; lane 2, PBL; lane 3, unbound fraction after MACS (C4B6⁻ cells); lane 4, bound fraction after MACS (C4B6⁺ cells); lane 5, salmon IgM. The 66 KDa band (*) in lane 3 and 4 is most likely BSA present in the MACS buffer. (C) qRT-PCR analysis of the C4B6⁻ cells. Gene expression of the following genes; IgM (B-cell marker), CD3, CD8 and TCRα (T-cell markers), MCSF-R (monocyte/macrophage marker), CD86 (involved in antigen presentation), CD83 (DC marker), MHC class II (APC), GATA-1 and G6F (thrombocyte/erythrocyte markers) is presented as mean normalized expression (MNE) using EF1α as reference gene (n = 4). The average of triplicates from four fish with standard error is shown. Note different scale in the inserted histogram.

Figure 3. The C4B6⁻ cells have potent phagocytic activity.

(A) A representative histogram showing the phagocytic activity (left panel) of the C4B6⁻ cells. Increased peak fluorescence indicates an increased number of ingested beads. The colors of the markers reflect the colors in the dot plot (middle panel). The dot plot shows the distribution of cells with various number of cells; cells without beads are shown as black dots, cells with one bead are red, cells with two beads are green, cells with three beads are blue and cells with more than three beads is shown as purple. Right panel, Diff Quick stained cells with various numbers of ingested beads. (B) TEM analyses of C4B6⁻ cells with beads. Left panel, a representative cell containing several 1 µm beads (magnification = 20 000×, scale bar = 1 µm). Middle panels, cells containing beads that are 2 µm (magnification = 15 000×, scale bar = 1 µm). Right panel is an enlargement of the delimited area of the middle, right panel showing beads that are just about to be ingested (magnification = 30 000×, scale bar = 0.5 µm). (C) SEM of C4B6⁻ cells ingesting beads that are 1 µm (two left panels) and 2 µm (two right panels). Notice the cell membrane that is about to enclose around the beads. A typical thrombocyte, containing one 2 µm bead, is shown in the right panel for comparison. In panels from left to right: Magnification (scale bar); 10 000× (1 µm), 30 000× (0.3 µm), 10 000× (1 µm) and 5000× (1 µm). (D). Different stages of bead capturing. Notice the veil (arrow). In panels from left to right: Magnification (scale bar); 8000× (1 µm), 5000× (1 µm) and 6500× (1 µm).

Figure 4. The C4B6⁻ cells have an oxygen-independent mechanism.

Panels to the left, C4B6⁺ cells, are included as positive controls. Panels to the right, C4B6⁻ cells. (A) Respiratory burst activity. Cells were stimulated with PMA and positive cells appear green in fluorescence microscopy due to RHO123 fluorescent (left panels). The corresponding bright field photos are shown in the right panels. Magnification: 400×. (B) Myeloperoxidase activity. Positive cells appear brown while cells without activity are blue. (C) Acid phosphatase staining. Granular sites of activity appear red. For the C4B6⁻ cells, notice that the overview picture to the right has less counterstaining, for easier observation of the positive granules. (D) Tartrate resistant acid phosphatase staining. Granular sites of activity appear red. Representative single cells are enlarged; PMN = polymorphonuclear, M = monocytes, L = leukocytes,* = representative C4B6⁻ cells, T = thrombocytes. Pictures in B–D are captured with 63× objective. Scale bar = 10 µm.

Figure 5. The C4B6⁻ cells change morphology upon stimulation with various mitogens.

Left panels, inverted microscope pictures. Middle panels, fluorescence microscopy pictures of cells stained with anti-TO antiserum, captured with 40× objective. Right panels, fluorescence microscopy pictures of cells stained with anti-TO antiserum, captured with 63× objective. (A) Cells without mitogen, 24 hrs incubation. (B) Cells stimulated with Con A and PMA for 18 hrs. (C) Cells stimulated with LPS for 24 hrs.